Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pK_as allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pK_a for Asp⁸ in the denatured state of wild-type, which is due to a nonnative interaction between Asp⁸ and Lys¹². Interestingly, the simulation also shows a nonnative interaction between Asp⁸ and Glu⁴⁸ in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape.     

Systematic analysis of ATG13 domain requirements for autophagy induction

Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.     

A randomized controlled trial of qigong for fibromyalgia

Introduction

Fibromyalgia is difficult to treat and requires the use of multiple approaches. This study is a randomized controlled trial of qigong compared with a wait-list control group in fibromyalgia.

Methods

One hundred participants were randomly assigned to immediate or delayed practice groups, with the delayed group receiving training at the end of the control period. Qigong training (level 1 Chaoyi Fanhuan Qigong, CFQ), given over three half-days, was followed by weekly review/practice sessions for eight weeks; participants were also asked to practice at home for 45 to 60 minutes per day for this interval. Outcomes were pain, impact, sleep, physical function and mental function, and these were recorded at baseline, eight weeks, four months and six months. Immediate and delayed practice groups were analyzed individually compared to the control group, and as a combination group.

Results

In both the immediate and delayed treatment groups, CFQ demonstrated significant improvements in pain, impact, sleep, physical function and mental function when compared to the wait-list/usual care control group at eight weeks, with benefits extending beyond this time. Analysis of combined data indicated significant changes for all measures at all times for six months, with only one exception. Post-hoc analysis based on self-reported practice times indicated greater benefit with the per protocol group compared to minimal practice.

Conclusions

This study demonstrates that CFQ, a particular form of qigong, provides long-term benefits in several core domains in fibromyalgia. CFQ may be a useful adjuvant self-care treatment for fibromyalgia.

Trial registration

clinicaltrials.gov NCT00938834.     

Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the (1)H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.     

Lipid-induced filamentous growth in Ustilago maydis

The phytopathogenic fungus Ustilago maydis is obligately dependent on infection of maize to complete the sexual phase of its life cycle. Mating interactions between haploid, budding cells establish an infectious filamentous cell type that invades the host, induces large tumours and eventually forms large masses of black spores. The ability to switch from budding to filamentous growth is therefore critical for infection and completion of the life cycle, although the signals that influence the transition have not been identified from the host or the environment. We have found that growth in the presence of lipids promotes a filamentous phenotype that resembles the infectious cell type found in planta. In addition, the ability of the fungus to respond to lipids is dependent on both the cAMP signalling pathway and a Ras/MAPK pathway; these pathways are known to regulate mating, filamentous growth and pathogenesis in U. maydis. Overall, these results lead us to hypothesize that lipids may represent one of the signals that promote and maintain the filamentous growth of the fungus in the host environment.     

Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.     

Transmembrane adaptor protein WBP1L regulates CXCR4 signalling and murine haematopoiesis

WW domain binding protein 1‐like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6‐RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non‐haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4‐family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l‐deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis.     

Yeast communities associated with stingless bees

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees were typical of species known to occur in flowers. Other yeast species found in adult bees were more typical of those found in the phylloplane. S. meliponinorum and the species in the C. apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied. Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.